Selenium is an essential micronutrient in the diet of humans and other mammals and many health benefits have been ascribed to this element including preventing cancer, heart disease and other cardiovascular and muscle disorders, inhibiting viral expression, delaying the progression of AIDS in HIV positive patients, slowing the aging process, and having roles in mammalian development, male reproduction and immune function. We proposed previously that the health benefits of selenium are due in large part to the presence of selenium in selenoproteins as the selenium-containing amino acid, selenocysteine (Sec). We previously established the biosynthetic pathway of Sec, the 21st amino acid in the genetic code, in eukaryotes and archaea and are now focusing our attention on the two Sec tRNA isoforms that we have shown are responsible for the synthesis of the two subclasses of selenoproteins designated housekeeping and stress-related selenoproteins; and on the methylase, designated Um34 methylase, that synthesizes the methyl group at the 2'-O-postion on the ribosyl moiety at nucleotide 34 of Sec tRNA. We previously provided strong evidence that addition of Um34 to the isoform, 5-methylcarobxymethyl-uridine (mcmU), to form 5-methylcarboxymethyl, 2'-O-methyluridine (mcmUm) requires that mcmU is aminoacylated with Sec, i.e., that the substrate for the methylase (designated Um34 methylase) which carries out this reaction is selenocysteyl-tRNA. In the past year, we completed and published our study involving the effect of the antibiotics, doxycycline (Dox), chloramphenicol (Cp) and geneticin (G418), on translation of thioredoxin reductase 1 (TR1), glutathione peroxidase 1 (GPx1) and glutathione peroxidase 4 (GPx4). This project was initially described in last year's annual report. We found that Dox, Cp and G418 interfered with insertion of selenocysteine (Sec), which is encoded by the stop codon, UGA, into selenoproteins in murine EMT6 cells. Treatment of EMT6 cells with these antibiotics reduced enzymatic activities and Sec insertion into thioredoxin reductase 1 (TR1) and glutathione peroxidase 1 (GPx1). However, these proteins were differentially affected due to varying errors in Sec insertion at UGA. In the presence of Dox, Cp or G418, the Sec-containing form of TR1 decreased, whereas the arginine-containing and truncated forms of this protein increased. We also detected antibiotic-specific misinsertion of cysteine and tryptophan. Furthermore, misinsertion of arginine in place of Sec was commonly observed in GPx1 and glutathione peroxidase 4. TR1 was the most and GPx1 the least affected by these translation errors. These observations were consistent with the differential use of two Sec tRNA isoforms and their distinct roles in supporting accuracy of Sec insertion into selenoproteins. The data reveal widespread errors in inserting Sec into proteins and in dysregulation of selenoprotein expression and function upon antibiotic treatment. A collaborative study with Dr. Vadim Gladyshev involved an examination of the location of the SECIS (SelenoCysteine Insertion Sequence) element in relation to the UGA Sec codon on selenoprotein expression. We observed that the location of UGA within the open reading frame of the corresponding selenoprotein is essential for efficient Sec insertion. UGA was found to sustain Sec insertion at the usual Sec positions, or in close positions relative to the natural UGA site, in TR1. Surprisingly, UGA was found to carry out Sec insertion effectively when UGA was moved to any inframe position in thioredoxin reductase 3 (TR3). Replacement of the 3'-UTR of TR3 with the corresponding segment from the ciliate, Euplotes crassus, wherein its TR restricts Sec insertion at the C-terminal region, demonstrated that the 3'-UTR of TR3 resulted in unrestricted Sec insertion into E. crassus TR. As expected, exchanges of 3'-UTRs between mammalian TR1 and E. crassus TR had no effect, since both proteins were shown to confer Sec insertion in the C-terminal region. Overall, the data suggest that mammals have the ability to restrict Sec insertion into normal positions within selenoproteins, but carry this out in a selenoprotein-specific manner, and that this process is controlled by the type of SECIS element in the 3'-UTR. In a collaborative study with Dr. Michael Howard, we used the recently developed technique of ribosome profiling to elucidate the effects of dietary selenium in combination with Sec tRNA modification on the translational mechanisms controlling selenoprotein synthesis in mouse liver. Dietary selenium levels were found to control gene-specific selenoprotein expression primarily at the translation level by differential regulation of UGA redefinition and Sec incorporation efficiency, although effects on translation initiation and mRNA abundance were also observed. Direct evidence that increasing dietary selenium causes a vast increase in ribosome density downstream of UGA-Sec codons for a subset of selenoprotein mRNAs and that the selenium-dependent effects on Sec incorporation efficiency are mediated in part by the degree of Sec-tRNA Um34 methylation. Furthermore, we provided evidence for translation in the 5'-UTRs for a subset of selenoproteins and for ribosome pausing near the UGA-Sec codon in those mRNAs encoding the selenoproteins most affected by selenium availability. The data demonstrate how dietary levels of the essential element selenium can alter the readout of the genetic code to affect the expression of an entire class of proteins. In collaboration with Dr. Ulrich Schweizer, a conditional knockout mouse encoding the SECIS binding protein 2 gene (abbreviated as Sbp2 or Secisbp2) was generated (S Seeher, T Atassi, Y Mahdi, BA Carlson, D Braun, EK Wirth, MO Klein, N Reix, AC Miniard, L Schomburg, DL Hatfield, DM Driscoll, U Schweizer: Secisbp2 is essential for embryonic development and enhances selenoprotein expression. [Submitted for publication]). Total knockout of Sbp2 was embryonic lethal. The targeted removal of Sbp2 in primary hepatocyte cells using RNAi technology resulted in greater down-regulation of selenoprotein mRNA abundance in SBP2-deficient hepatocytes than in the corresponding Sec tRNA-deficient cells. Interestingly, despite enormous reductions in deiodinase 1 (Dio1) and selenoprotein P (Sep1) mRNAs in SBP2-deficient liver, low but significant expression of selenoproteins were found that was not observed in liver lacking Sec tRNA . The data suggest that 1) SBP2 normally provides protection of selenoprotein mRNAs from degradation by its interaction with these mRNAs and 2) provide further evidence that Sec tRNA is indeed the central component of selenoprotein synthesis as its loss results in total loss in translation of this class of selenium-containing proteins, while the loss of other essential components (e.g., SBP2) still maintains some selenoprotein synthesis.